Kit for predicting implantation success in assisted fertilization

ABSTRACT

A kit for determining for a female subject the implantation potential of embryos obtained or to be obtained by assisted fertilization is described. The kit includes at least one reagent suitable for detection of levels of FF G-CSF or FF G-CSF mRNA, such as anti-G-CSF antibody or a nucleic acid probe for detection of levels of G-CSF mRNA. The kit may also include a set of concentration standards of FF G-CSF and aspirator tips for removing an oocyte and follicular fluid.

This application is a continuation-in-part of U.S. application Ser. No.12/374,472, filed Jan. 20, 2009 which is incorporated herein byreference and which is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application PCT/EP2007/057430, filed Jul. 18, 2007, whichclaims priority to U.S. Provisional Application No. 60/832,094, filedJul. 21, 2006.

Assisted fertilization, such as in vitro fertilization (IVF) orintracytoplasmic sperm injection (ICSI) has been used in human patientswith infertility problems successfully for three decades. Despiteextensive research it is still a difficult and expensive procedure and alow implantation rate per transferred embryos (15-20%) is generallyobserved.

Hospitals and private centers providing an assisted fertilizationservice, base their selection after fertilization of the oocyte oncharacteristics of the embryo so produced. For example, selection may bebased on the morphology of the embryo (Guerif F et al., 2007, Hum Reprod22(7):1973), or on the production of soluble HLA-G by the embryos (FuzziB, et al., 2002, Eur J Immunol. February; 32(2):311-5.). Both thesetechniques require interference with the embryo.

To increase the pregnancy success, the number of embryos transferredtypically more than one. In Europe, it is normal practice to transfertwo embryos to the uterine cavity. In the US, it is more, usually threeor four embryos are transferred. The adverse effect of such a policy isto increase the number of multiple pregnancies and the subsequentrelated obstetrical pathologies, such as prematurity and low birth ratemainly.

Furthermore, assisted fertilization is an expensive procedure and canalso be psychologically traumatic for a patient. Surgical procedures arerequired to collect eggs for assisted fertilization and followingfertilization, further surgery is required to implant fertilized eggs inthe womb. The recipient must then wait for a period of time before itcan be determined whether or not pregnancy has been established. In somecases, pregnancy may never be established despite repeated attempts andthese cases represent a considerable expense to society, both infinancial and human terms.

Therefore, it would be desirable to provide an assay and kit which canindicate the potential for implantation of an oocyte beforefertilization, enabling the chances of successful implant of the embryoto be maximized, and allowing indications of low success rates to beused to avoid the abovementioned trauma and costs of assistedfertilization.

FIGURE LEGENDS

FIG. 1. ROC curve from a Luminex experiment to detect FF G-CSF using aBiorad Luminex kit. The true positive rate (Sensitivity) is plotted infunction of the false positive rate (100-Specificity) for differentcut-off points of FF G-CSF concentration. Each point on the ROC plotrepresents a sensitivity/specificity pair corresponding to a particulardecision threshold. The area under the ROC curve is a measure of howwell FF G-CSF can distinguish between two main diagnostic groups(certain implantation/no implantation). Line 1: The Area under curve is0.82, indicating that a randomly selected individual from the positivegroup has a test value larger than that for a randomly chosen individualfrom the negative group in 82% of the time. Line 2: Area under the ROCcurve is 0.5 representing the null hypothesis.

FIG. 2. ROC curve from a Luminex experiment to detect FF G-CSF using anR and D Luminex kit. Line 1: The Area under curve is 0.72, indicatingthat a randomly selected individual from the positive group has a testvalue larger than that for a randomly chosen individual from thenegative group in 72% of the time. Line 2: Area under the ROC curve is0.5 representing the null hypothesis.

FIG. 3. Graph showing the variation in concentration of individualfollicular fluid of a same cohort of embryos obtained from multiplesubjects. Each box shows the variation of individual follicular fluidsfrom the mean in a same cohort of embryos generated.

SUMMARY OF THE INVENTION

Embodiments of the invention are based on an unexpected finding by theinventors that a female subject providing a plurality of oocytes underovarian hyperstimulation will exhibit a variation in the levels ofseveral cytokines and growth factors present in the follicular fluid ofthe follicle from which each oocyte is derived. Furthermore, theinventors found there is a strong correlation between a high level ofgranulocyte-colony stimulating factor (G-CSF) present in the follicularfluid of the individual follicle from which an oocyte is derived and ahigh implantation potential of an embryo obtained by fertilization ofsaid oocyte. It has never been demonstrated before that, for the samesubject, the follicular fluid surrounding each individual oocyte mayvary in composition, and that said composition is indicative of thesuccess of implantation of the subsequently fertilized oocyte. Thisfinding allows a plurality of embryos obtained from a single patient tobe ranked in order of implantation potential. For the first time,patients showing a borderline fertility potential using indicators thataverage oocyte fertility markers (e.g. 11-beta HSD) may be found to haveoocytes showing a high implantation potential against a poor overallaverage; this offer new possibilities for previouslyinfertile-indicating females. Furthermore, the method offers thepossibility to rate each oocyte individually and thus embryoindividually, without interference to the embryo or oocyte.

Embodiments of the present invention relate to an assay for determiningthe implantation potential of a plurality of embryos each obtained or tobe obtained by assisted fertilization of an oocyte of a female subject,comprising measuring the levels of G-CSF in the follicular fluid presentin the follicle from which each oocyte is derived, and determining theimplantation potential of each embryo from the level of follicular fluidG-CSF. The oocyte from the follicle with the highest level of G-CSF infollicular fluid gives rise to an embryo with the greatest implantationpotential.

Embodiments of the present invention relates to an assay kit which canbe used to predict the outcome of assisted fertilization in a femalepatient. The invention also relates to such assay and kit for use in amethod of fertilization treatment, to improve implantation.

One embodiment of the invention is an assay for determining for a femalesubject the implantation potential of embryos obtained or to be obtainedby assisted fertilization comprising:

(i) measuring, for a plurality of oocytes collected from said subject,the level of follicular fluid granulocyte-colony stimulating factor(G-CSF) present in the follicular fluid (FF) of a follicle of eachcollected oocyte; and

(ii) determining from the levels of FF G-CSF measured, the implantationpotentials of the embryos obtained or to be obtained by assistedfertilization of the oocytes.

Another embodiment of the invention is an assay as described above,wherein oocytes having the highest levels of FF G-CSF have the highestpotential of implantation.

Another embodiment of the invention is an assay as described above,wherein each sample of FF is obtained from a follicular aspirate.

Another embodiment of the invention is an assay as described above,wherein respective levels of FF G-CSF are measured within 20 hours ofcollection of the follicular aspirate.

Another embodiment of the invention is an assay as described above,wherein a level of FF G-CSF equal to or less than 20.6 pg/ml determinesno or a low potential of implantation.

Another embodiment of the invention is an assay as described above,wherein a level of FF G-CSF equal to or greater than 24.0 pg/mldetermines a high potential of implantation.

Another embodiment of the invention is an assay as described above,wherein the respective levels of FF G-CSF are measured using animmunoassay.

Another embodiment of the invention is an assay as described above,wherein the respective levels of FF G-CSF are measured using an acompetitive or immunometric assay, such as RIA, IRMA, ELISA, or ELISPOTassay.

Another embodiment of the invention is an assay as described above,wherein the respective levels of FF G-CSF are measured using a Luminexassay.

Another embodiment of the invention is an assay as described above,wherein the Luminex assay employs a Biorad or R and D Luminex Kit.

Another embodiment of the invention is an assay as described above,wherein the respective levels of FF G-CSF are measured by determiningthe levels of FF G-CSF mRNA.

Another embodiment of the invention is an assay as described above,wherein the respective levels of FF G-CSF are measured by any of surfacePlasmon resonance, fluorescence resonance energy transfer,bioluminescence resonance energy transfer, fluorescence quenchingfluorescence, fluorescence polarization, MS, HPLC, HPLC/SM, HPLC/MS/MS,capillary electrophoresis, rod or slab gel electrophoresis.

Another embodiment of the invention is a kit for use in performing theassay as described above, comprising at least one reagent suitable fordetection of levels of FF G-CSF or FF G-CSF mRNA.

Another embodiment of the invention is a kit as described above, furthercomprising a set of concentration standards of FF G-CSF.

Another embodiment of the invention is a kit as described above, furthercomprising a plurality of aspirator tips for removing an oocyte andfollicular fluid from a subject.

Another embodiment of the invention is a method for assistedfertilization of a female subject comprising:

(i) collecting a plurality of oocytes from said subject,

(ii) determining the implantation potential for an embryo derived fromeach oocyte according to the assay as described above,

(iii) fertilizing the oocytes corresponding to embryos having a highpotential for implantation, and

(iv) implanting the embryo so obtained into the female subject.

Another embodiment of the invention is a method for assistedfertilization of a female subject comprising:

(i) collecting a plurality of oocytes from said subject,

(ii) determining the implantation potential for an embryo derived fromeach oocyte according to the assay as described above,

(iii) fertilizing the oocytes to obtain embryos, and

(iv) implanting the embryos having a high implantation potential.

Another embodiment of the invention is a kit for determining for afemale subject the implantation potential of embryos obtained or to beobtained by assisted fertilization comprising at least one reagentsuitable for detection of levels of FF G-CSF or FF G-CSF mRNA. The kitmay further comprise a set of concentration standards of G-CSF. Theconcentration standards may be commercial G-CSF standards. The kit mayfurther comprise a plurality of aspirator tips for removing an oocyteand follicular fluid from a subject. At least one reagent may comprisean anti-G-CSF antibody for detection of levels of FF G-CSF; theanti-G-CSF antibody may be labeled. At least one reagent may comprise anucleic acid probe for detection of levels of G-CSF mRNA; the nucleicacid probe may be labeled. The at least one reagent may comprise aspecific antibody fragment, nanobody, affibody, an aptamer, aphotoaptamer, a small molecule, an interacting partner, a specificallybinding protein or peptide, a Darpin, or an ankyrin, an isotopicallylabeled tracer, or a ligand, specifically binding to said G-CSF. Thespecific antibody fragment may be a Fab fragment, an Fv fragment, or ascFv fragment. The at least one reagent may comprise a specific labeledantibody fragment, a labeled nanobody, a labeled affibody, a labeledaptamer, a labeled photoaptamer, a labeled small molecule, a labeledinteracting partner, a labeled specifically binding protein or peptide,a labeled Darpin, a labeled ankyrin, or a labeled ligand, specificallybinding to said G-CSF. The specific labeled antibody fragment may be alabeled Fab fragment, a labeled Fv fragment, or a labeled scFv fragment.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart. All publications referenced herein are incorporated by referencethereto. All United States patents and patent applications referencedherein are incorporated by reference herein in their entirety includingthe drawings.

The articles “a” and “an” are used herein to refer to one or to morethan one, i.e. to at least one of the grammatical object of the article.By way of example, “a sample” means one sample or more than one sample.

The recitation of numerical ranges by endpoints includes all integernumbers and, where appropriate, fractions subsumed within that range(e.g. 1 to 5 can include 1, 2, 3, 4 when referring to, for example, anumber of samples, and can also include 1.5, 2, 2.75 and 3.80, whenreferring to, for example, concentrations). The recitation of end pointsalso includes the end point values themselves (e.g. from 1.0 to 5.0includes both 1.0 and 5.0)

As mentioned elsewhere present invention relates to an unexpectedfinding by the inventors that a female subject providing a plurality ofoocytes under ovarian hyperstimulation will exhibit a variation in thelevels of several cytokines and growth factors present in the follicularfluid of the follicle from which each oocyte is derived. Furthermore,the inventors found there is a strong correlation between a high levelof granulocyte-colony stimulating factor (G-CSF) present in thefollicular fluid of the individual follicle from which an oocyte isderived and a high implantation potential of an embryo obtained byfertilization of said oocyte. It has never been demonstrated beforethat, for the same subject, the follicular fluid surrounding eachindividual oocyte may vary in composition, and that said composition isindicative of the success of implantation of the subsequently fertilizedoocyte. This finding allows a plurality of embryos obtained from asingle patient to be ranked in order of implantation potential. For thefirst time, patients showing a borderline fertility potential usingindicators that average oocyte fertility markers (e.g. 11-beta HSD) maybe found to have oocytes showing a high implantation potential against apoor overall average; this offer new possibilities for previouslyinfertile-indicating females. Furthermore, the method offers thepossibility to rate each oocyte individually and thus embryoindividually, without interference to the embryo or oocyte.

Embodiments of the present invention thus relate to an assay method andassay kit which can be used to predict the outcome of assistedfertilization in a female patient. Embodiments of the invention alsorelate to such assay and kit for use in a method of fertilizationtreatment, to improve implantation. Although our invention describedbelow has been developed from research on human female patients, it willbe applicable to any mammalian female and can be used to increase thesuccess of, for example, captive breeding programs of endangered speciesor commercial breeding by assisted fertilization of livestock such ascattle or horses. Preferably the subject has undergone fertilitypretreatment (e.g. ovarian hyperstimulation) to increase the number ofeggs produced per monthly cycle. Assisted fertilization, as used herein,refers to ex vivo fertilization methods where the oocyte is fertilizedoutside the female body, such as in vitro fertilization (IVF) orintracytoplasmic sperm injection (ICSI).

One embodiment of the invention is an assay for determining theimplantation potential of a plurality of embryos each obtained or to beobtained by assisted fertilization of an oocyte of a female subject,comprising measuring the levels of G-CSF in the follicular fluid presentin the follicle from which each oocyte is derived, and determining theimplantation potential of each embryo from the level of follicular fluidG-CSF.

Another embodiment of the present invention is an assay for determiningfor a female subject the implantation potential of embryos obtained orto be obtained by assisted fertilization comprising:

(i) measuring, for a plurality of oocytes collected from said subject,the level of follicular fluid granulocyte-colony stimulating factor(G-CSF) present in the follicular fluid (FF) of a follicle of eachcollected oocyte; and

(ii) determining from the levels of FF G-CSF measured, the implantationpotentials of the embryos obtained or to be obtained by assistedfertilization of the oocytes.

The oocyte from the follicle with the highest level of G-CSF infollicular fluid gives rise to an embryo with the greatest implantationpotential.

Granulocyte colony stimulating factor (G-CSF) is a naturally generatedcytokine belonging to the family of hemopoietic growth factor (Clark, etal., 1987, Science 236(4806):1229). Its main role described is to act onproliferation, differentiation, and activation of hematopoietic cells ofthe neutrophilic lineage (Mielcarek et al., 1996, Blood 87(2):574,Visani et al., 1995, 18(5-6):423). Primarily produced by hemopoieticcells, G-CSF is also produce by non-hemopoietic cells, such as in thereproduction tract: the human luteinized follicular granulosa cells(Salmassi A, at al, 2004, Fertil Steril, 81 Suppl 1:786.), endometrialcells (Giacomini G, et al., 1995, Hum Reprod 10(12):3259.), decidua andplacenta (Duan J. S., 1990, Osaka City Med J 36(2):81; Miyama M at al.,1998, Osaka City Med J., 44(1):85) and various fetal tissue (Calhoun atal., 1999. Pediatr Res 46(3):333). In the ovary, G-CSF protein and itsreceptor were located (western blot and immunohistochemistry) mainly ingranulosa cells of the follicle and luteal cells (Salmassi, at al.,2004).

The level of follicular fluid G-CSF (FF G-CSF) is preferably measuredwithin the day of oocyte collection. As is known to the skilled person,follicular aspiration is guided using transvaginal sonography afterlocal or general anesthesia. Each follicular fluid corresponding to oneovarian follicle visualized through vaginal sonography is aspiratedindividually. The capture of each oocyte does not require any othermanipulation because the follicular fluid, which surrounds the oocyte,is aspirated along with the oocyte. Inspection of the follicular fluidunder microscope allows immediate identification of the presence of theoocyte. Instead of pooling the follicular fluids and respective oocytes,the oocyte is separated at the time of collection so the levels of FFG-CSF can be individually measured. According to one aspect of theinvention, the level of FF G-CSF is measured with 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours of oocytecollection, of within a time between any two of the aforementionedvalues. Preferably the level of FF G-CSF is measured within 1 to 20hours of oocyte collection.

The level of FF G-CSF associated with an oocyte can be measured usingany suitable quantitative assay. The measuring may be performed, forexample, using a method selected from biochemical assay (e.g., solid orliquid phase immunoassay), surface plasmon resonance, fluorescenceresonance energy transfer, fluorescence quenching, and fluorescencepolarization. Such techniques are well known in the art and are brieflydescribed herein below.

Biochemical assays generally rely on the immobilization of an analytecomponent, for example, to a membrane or other solid support, andexposure to a ligand. After washing away excess ligand, bound ligand isdetected by immunoassay, or by using labeled ligand (e.g., radio-labeledligand, fluorescently labeled ligand, particulate labeled ligand etc.).

Methods to determine and obtain ligands which bind with high affinity toa specific analyte in are also available in the art; see for exampleWO89/09088 entitled “Paralog Affinity Chromatography”. In an example ofan immunoassay, antibodies against G-CSF may be immobilized ontomagnetic beads and exposed to a sample of follicular fluid. Bound G-CSFcan be detected using primary and secondary antibody immunoassays toarrive at a concentration. Typically, an immunoassay is calibrated useda set of standards. Solid phase immunoassays are described for examplein U.S. Pat. No. 4,376,110. Variations of the immunoassays within thescope of the invention include any competitive or immunometric assayformat using anti-G-CSF antibodies, for instance RIA(radio-immunoassay), ELISA (enzyme-linked immunosorbent assay), ELISPOT(enzyme-linked immunosorbent spot) or Luminex (bead-based multiplexsandwich immunoassay).

The levels FF G-CSF are preferably measured by using Luminex technology.Luminex is a highly sensitive method for measuring simultaneously thelevels of specific components in a system. It makes use of solid phase,colour (dye) coded microspheres that are small enough to behave almostas a solution in a liquid. Each microsphere is coated with an antibody,or other ligand-binding reagent specific for the detected components(e.g. FF G-CSF). The components of the sample are captured and detectedon the microspheres. Within an analyzer, lasers excite the internal dyesthat identify each microsphere particle, and also any reporter dyecaptured during the assay. Many readings are made on each bead set, tovalidate the results. In this way, a sensitive multiplex assay is madethat is both rapid and precise. Preferably, the levels of FF G-CSF aremeasured using a kit(s) manufactured by Biorad® or R and D®. In apreferred embodiment the Biorad® kit is the Human Cytokine FluorescentBead Immunoassay Assay Kit, Bio-Plex™ (Hercules, Calif., USA, 17A11127).In another preferred embodiment, the R and D kit is the LUH000, LUH279,LUH270, LUH271, LUH278, LUH208, LUH214, LUH215B, LUH285, LUH200, LUH280,LUH201, LUH202, LUH204, LUH205, LUH206, LUH217, LUH317, LUH210, LUH293,LUB000, LUB320, LUB294, LUB219, and/or LUB213 kit.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes monoclonal antibodies, polyclonalantibodies, and fragments of whole antibodies which retain their bindingactivity for a target antigen. Such fragments include Fv, F(ab′) andF(ab′)2 fragments, as well as single chain antibodies. Furthermore, theantibodies and fragments thereof may be humanized antibodies, e.g. asdescribed in EP-A-239400 (Winter).

Antibodies against FF G-CSF may be monoclonal or polyclonal antibodies.Monoclonal antibodies may be prepared by conventional hybridomatechnology using the proteins or peptide fragments thereof, as animmunogen. Polyclonal antibodies may also be prepared by conventionalmeans which comprise inoculating a host animal, for example a rat or arabbit, with a peptide of the invention and recovering immune serum.

Alternatively, levels of FF G-CSF may be estimated by analyzing thelevels of FF G-CSF mRNA in the granulosa cells. Granulosa cells aroundthe corona radiata may be stored at the stage of the decoronisation ofeach oocytes and be stored in RNA stabiliser (e.g. at 80° C.) untilassay. Probes for the FF G-CSF gene may be designed for use as probes,for example for use in a nucleic acid (PCR) amplification assay and/orhybridization. Methods and conditions for performing a PCR andhybridization reactions are known in the art, and can be found, forexample, in Molecular Cloning: A Laboratory Manual (Third Edition)(Joseph Sambrook, Peter MacCallum, David Russell, Cold Spring HaborLaboratory Press) or could be performed by a quantigene plex assay,which is designed to quantitate multiple target-specific RNA molecules(Panomics).

A surface plasmon resonance assay may, alternatively, be used as aquantitative method to measure the level of G-CSF in a follicular fluidsample. A chip-bound anti-G-CSF antibody is challenged with a follicularfluid and the surface plasmon resonance measured. Binding reactions areperformed using standard concentrations to arrive at the levels of G-CSFof G-CSF in the follicular fluid.

FRET (fluorescence resonance energy transfer) may also be used tomeasure the level of G-CSF in a follicular fluid sample. The G-CSF andanti-G-CSF antibody are labeled with a complementary pair of donor andacceptor fluorophores. While bound closely together by theG-CSF:anti-G-CSF antibody interaction, the fluorescence emitted uponexcitation of the donor fluorophore will have a different wavelengththan that emitted in response to that excitation wavelength when theG-CSF and anti-G-CSF antibody are not bound, providing for quantitationof bound versus unbound molecules by measurement of emission intensityat each wavelength. Binding reactions can be compared with a set ofstandards to arrive at the level of G-CSF in the follicular fluid.

BRET (bioluminescence resonance energy transfer) may also be used tomeasure the level of G-CSF in a follicular fluid sample. Light isemitted by an acceptor when in close proximity to the donor, i.e., whena G-CSF: anti-G-CSF antibody interaction complex is formed. By comparingthe interaction with a set of standards, the level of G-CSF in thefollicular fluid is determined.

Fluorescence quenching fluorescence similarly provides a measurement ofG-CSF levels. Generally, a decrease in fluorescence of the labeledanti-G-CSF antibody is indicative that the G-CSF bearing the quencherhas bound. Of course, a similar effect would arise when a G-CSF isfluorescently labeled and anti-G-CSF antibody bears the quencher. Bycomparing the interaction with a set of standards, the level of G-CSF ina follicular fluid sample can be measured.

Fluorescence polarization measurement can also determine the level ofG-CSF in a follicular fluid sample. Complexes, such as those formed byG-CSF associating with a fluorescently anti-G-CSF antibody, would havehigher polarization values than uncomplexed, labelled anti-G-CSFantibody. This form the basis for determining the levels of G-CSF in afollicular fluid sample, which measurements are typically performedconcurrent with a set of standard G-CSF concentrations.

Other methods that can be used for quantitatively assaying G-CSF in theFF include mass spectrometry (MS), high performance liquidchromatography (HPLC), HPLC/MS, HPLC/MS/MS, capillary electrophoresisand rod or slab gel electrophoresis associated with image analysis. Suchtechniques are well known in the art as described, for example, inModern HPLC for Practicing Scientists (Dong, M, Wiley-Interscience, June2006), Tandem Mass Spectrometry (McLafferty F. W. John Wiley & Sons Inc,November, 1983), Mass Spectrometry for Biotechnology (Siuzdak, G.,Academic Press, February 1996), Clinical Applications of CapillaryElectrophoresis (Methods in Molecular Medicine) (Palfrey S. M., HumanaPress, June 1999), Handbook of Capillary Electrophoresis, SecondEdition, (Landers J. P. CRC; December 1996), High-ResolutionElectrophoresis and Immunofixation: Techniques and Interpretation(Keren, D. F. Hodder Arnold, January, 1994).

Once the levels of FF G-CSF have been measured in a plurality of oocytesfrom a single patient, the results may be used to establish the relativeimplantation potential of embryos obtained by fertilization of saidoocytes i.e. a ranking order. The level of FF G-CSF may be used todetermine whether all, some or none of the oocytes will afterfertilization establish implantation in a female subject undergoingassisted fertilization treatment. In addition, the level of FF G-CSF maybe used to determine whether all, some or none of the embryos willimplant in a female subject undergoing assisted fertilization treatment.

In our studies, we have measured levels of FF G-CSF using immunoassays,in particular using Luminex technology from Biorad and R&D. We havefound that those embryos derived from oocytes having a concentration ofFF G-CSF equal to or less than 20.0 pg/ml show a reduced or noimplantation success. In contrast, embryos derived from oocytes having aconcentration of FF G-CSF above 24 pg/ml show a certain implantation.

Those of skill in the art will appreciate that although in our researchwe have determined a “threshold” level of FF G-CSF below which embryosare not implanted (and above which patients have significantly improvedprobability of implantation), the value is a statistical measure andother measurements and thresholds can be used. In practicing theinvention, it is most important to achieve consistency of assay, and soeach individual practitioner (or assisted fertilization team) will becapable of establishing their own particular assay method anddetermining their own threshold level. This could be established byfirst conducting a historical study on samples from previous patients.

Thus, the level of FF G-CSF mentioned above represents the measure wehave used in our studies as a suitable limit. However, if levels of FFG-CSF were to be measured in any of the other ways mentioned above, itwould be desirable to conduct, using routine procedures, a control usingour method of assay in order to determine the relationship between ourresults and the results of other methods, in order to make directcomparisons.

According to one aspect of the invention, an embryo derived from anoocyte where a level of FF G-CSF in its follicle is equal to or lessthan 21.6 pg/ml, 21.4 pg/ml, 21.2 pg/ml, 21.0 pg/ml, 20.8 pg/ml, 20.6pg/ml, 20.4 pg/ml, 20.2 pg/ml, 20.0 pg/ml, 19.8 pg/ml, 19.6 pg/ml, 19.4pg/ml, 19.2 pg/ml, 19.0 pg/ml, 18.8 pg/ml, 18.6 pg/ml, 18.4 pg/ml, 18.2pg/ml, 18.0 pg/ml 17.8 pg/ml, 17.6 pg/ml, 17.4 pg/ml, 17.2 pg/ml, 17.0pg/ml, 16.8 pg/ml, 16.6 pg/ml, 16.4 pg/ml, 16.2 pg/ml, 16.0 pg/ml, 15.8pg/ml, 15.6 pg/ml, 15.4 pg/ml, 15.2 pg/ml, 15.0 pg/ml or a level betweenany two of the aforementioned values, is predicted to have a lowimplantation potential. Preferably, a level of FF G-CSF equal to or lessthan 15.0 pg/ml to 20.0 pg/ml, more preferably equal to or less than19.8 to 20.6 pg/ml, most preferably less than 20.6 pg/ml is predicted tohave no or a low implantation potential. The levels of this embodimentare considered threshold levels for a method of assisted fertilization(below). A low level of implantation is a probability of implantation of10%, 9%, 8% or less.

According to one aspect of the invention, an embryo derived from anoocyte where a level of FF G-CSF in its follicle is equal to or lessthan 34.0 pg/ml, 33.5 pg/ml, 33.0 pg/ml, 32.5 pg/ml, 32.0 pg/ml, 31.5pg/ml, 31.0 pg/ml, 30.5 pg/ml, 30.0 pg/ml, 29.5 pg/ml, 29.0 pg/ml, 28.5pg/ml, 28.0 pg/ml, 27.5 pg/ml, 27.0 pg/ml, 26.5 pg/ml, 26.0 pg/ml, 25.5pg/ml, 25.0 pg/ml, 24.5 pg/ml, 24.0 pg/ml, 23.5 pg/ml, 23.0 pg/ml, 22.5pg/ml, 22.0 pg/ml, 21.5 pg/ml, 21.0 pg/ml, 20.5 pg/ml, 20.0 pg/ml, 19.5pg/ml, 19.0 pg/ml, 18.5 pg/ml, 18.0 pg/ml, 17.5 pg/ml, 17.0 pg/ml, 16.5pg/ml, 16.0 pg/ml, 15.5 pg/ml, 15.0 pg/ml or a level between any two ofthe aforementioned values, is predicted to have a likely implantationsuccess. Preferably, a level of FF G-CSF in the range 15.0 pg/ml to 34.0pg/ml, more preferably in the range 20.0 to 24.0 pg/ml predicted to belikely to be implanted. Likely to be implanted means a higher chance ofsuccess that no certainty of implantation; a likely potential ofimplantation means a probability of implantation of 15% to 25%. Thelevels of this embodiment are considered threshold levels for a methodof assisted fertilization (below).

According to one aspect of the invention, an embryo derived from anoocyte where a level of FF G-CSF in its follicle is equal to or higherthan 22.0 pg/ml, 22.1 pg/ml, 22.2 pg/ml, 22.3 pg/ml, 22.4 pg/ml, 22.5pg/ml, 22.6 pg/ml, 22.7 pg/ml, 22.8 pg/ml, 22.9 pg/ml, 23.0 pg/ml, 23.1pg/ml, 23.2 pg/ml, 23.3 pg/ml, 23.4 pg/ml, 23.5 pg/ml, 23.6 pg/ml, 23.7pg/ml, 23.8 pg/ml, 23.9 pg/ml, 24.0 pg/ml, 24.1 pg/ml, 24.2 pg/ml, 24.3pg/ml, 24.4 pg/ml, 24.5 pg/ml, 24.6 pg/ml, 24.7 pg/ml, 24.8 pg/ml, 24.9pg/ml, 25.0 pg/ml, 25.1 pg/ml, 25.2 pg/ml, 25.3 pg/ml, 25.4 pg/ml, 25.5pg/ml, 25.6 pg/ml, 25.7 pg/ml, 25.8 pg/ml, 25.9 pg/ml, 26.0 pg/ml, 26.1pg/ml, 26.2 pg/ml, 26.3 pg/ml or a level between any two of theaforementioned values, is predicted to have a high implantationpotential. Preferably, a level of FF-CSF equal to or higher than 24.0pg/ml, more preferably higher than 35 pg/ml is predicted to have a highimplantation potential. The levels of this embodiment are consideredthreshold levels for a method of assisted fertilization (below). A highlevel of implantation is a probability of implantation of 30%, 35%, 40%,43%, 44% or more.

If levels of FF G-CSF in such patients is significantly below the levelassociated with likely or certain implantation in all collected oocytes,then there would be a saving in time, money and stress to the patientnot to undertake implantation. In such cases, it will be possible forthe practitioner (or assisted fertilization clinic) to decide whether ornot to even attempt a first implantation. On the other hand, if one ormore oocytes indicate a high or complete certainty of implantation,these oocytes alone may be fertilized and the embryos so obtainimplanted, so saving money and resources by fertilizing only thoseoocytes likely to become established as embryos. Alternatively, all theoocytes may be fertilized, and only those embryos derived from oocytesindicating a high or complete certainty of implantation, are implanted;this allows a higher chance of success as the indication of implantationdoes not necessarily correlated with chances of fertilization.

The present invention significantly increase the implantation rate whiledecreasing the number of embryos replaced. It also allows a specialistto become more efficient in preventing multiple pregnancies and all therelated fetal and maternal morbidity. The oocyte and thus the embryowith the highest potential can be implanted implant, therefore, allows apolicy of single embryo transfer while not decreasing the overallpregnancy rate.

The assay described herein may also be employed in a method of assistingthe fertilization of a female subject. One embodiment of the inventionis a method for assisted fertilization of a female subject comprising:

(i) collecting a plurality of oocytes from said subject,

(ii) determining the level of FF G-CSF in the follicle of each collectedoocyte,

(iii) fertilizing the oocytes having the highest FF G-CSF levels, and

(iv) implanting the embryo so obtained into the female subject.

The number of oocytes subjected to further fertilization may be 1, 2, 3,4 or 5 or more. Alternatively, 50%, 40%, 30%, 20%, or 10% of the oocytesare fertilized, which percentage have the highest levels of FF G-CSF.

Another embodiment of the invention is a method for assistedfertilization of a female subject comprising:

(i) collecting a plurality of oocytes from said subject,

(ii) determining the level of FF G-CSF in the follicle of each collectedoocyte,

(iii) fertilizing the oocytes to obtain embryos,

(iv) implanting the embryos derived from oocytes having the highest FFG-CSF levels.

The number of embryos implanted may be 1, 2, 3, 4 or 5 or more.Alternatively, 50%, 40%, 30%, 20%, or 10% of the embryos are implanted,which percentage have the highest levels of FF G-CSF.

Another embodiment of the invention is a method for assistedfertilization of a female subject comprising:

(i) collecting a plurality of oocytes from said subject,

(ii) determining the level of FF G-CSF in the follicle of each collectedoocyte,

(iii) fertilizing the oocytes to obtain embryos,

(iv) implanting the embryos derived from oocytes having a FF G-CSF levelabove a predetermined threshold.

The number of embryos implanted may be 1, 2, 3, 4 or 5 or more.Alternatively, 50%, 40%, 30%, 20%, or 10% of the embryos are implanted,which percentage have the highest levels of FF G-CSF.

Another embodiment of the invention is a method for assistedfertilization of a female subject comprising:

(i) collecting a plurality of oocytes from said subject,

(ii) determining the implantation potential for an embryo derived fromeach oocyte according to the assay as defined above,

(iii) fertilizing the oocytes corresponding to embryos having a highpotential for implantation, and

(iv) implanting the embryo so obtained into the female subject.

Another embodiment of the invention is a method for assistedfertilization of a female subject comprising:

(i) collecting a plurality of oocytes from said subject,

(ii) determining the implantation potential for an embryo derived fromeach oocyte according to the assay as defined above,

(iii) fertilizing the oocytes to obtain embryos, and

(iv) implanting the embryos having the highest implantation potential.

The embodiments described above in regard of the assay apply tocorresponding embodiments of the method of assisted fertilization. Thethreshold values are indicated elsewhere herein. The skilled person willunderstand that intervening steps may be present such as freezing afteroocyte collection. By use of the present invention, it will be possiblefor assisted fertilization clinics to allocate resources moreefficiently, so that patients with low levels of FF G-CSF in thefollicle of a recovered oocyte who are unlikely to become pregnant byassisted fertilization treatment are not treated.

Kits for use in performing the assay of the invention may be provided.Such kits include at least one reagent useful for the detection of FFG-CSF.

Suitable reagents include one or more of a specific antibody fragment,nanobody, affibody, an aptamer, a photoaptamer, a small molecule, aninteracting partner, a specifically binding protein or peptide, aDarpin, or an ankyrin, an isotopically labeled tracer, or a ligand,specifically binding to said G-CSF, optionally be linked to a label. Thespecific antibody fragment is a Fab fragment, an Fv fragment, or a scFvfragment.

The reagents and their preparation are well known in the art, and do notrequire elaboration. Nonetheless, for the sake of completeness, a briefsummary of some of the reagents is provided. A nanobody is asingle-domain antibody having a single monomeric variable antibodydomain able to bind selectively to a specific antigen. A specificallybinding nanobody may be prepared from a library of engineered nanobodieshaving a different binding regions, screened against a target. Phagedisplay technology may be employed to select binding recombinantstrains. Suitable construction and screening techniques are mentioned inWO 94/04678. An affibody is an engineered binding molecule having highstability and specificity, containing three alpha helices as a proteinscaffold. The were first described in Nord, K; Nilsson, J; et al (1995).“A combinatorial library of an α-helical bacterial receptor domain”.Prot. Eng. 8 (6): 601-608. A specifically binding affibody may beprepared from a library of engineered affibody having a differentbinding region, screened against a target. Phage display technology maybe employed to select binding recombinant strains. A Darpin (designedankyrin repeat proteins) is an engineered specific binding proteincontaining a number of repeats (e.g. 2, 3, 4, 5 6 or more) of an ankyrinsubunit. A specifically binding Darpin may be prepared from a library ofengineered Darpins having a different binding region, screened against atarget. Phage display technology may be employed to select bindingrecombinant strains. Suitable construction and screening techniques arementioned in WO 02/20565 and WO 2010/060748. Aptamers are engineeredoligonucleic acid or peptide molecules typically having a scaffold andvariable binding domain, that binds to a specific target molecule; aswith others they may be screened against a target using phage displaytechnology.

Suitable reagents include antibodies, or other suitable ligand-bindingreagents, against FF G-CSF optionally linked to a label. Typical labelsare those commonly used in immunoassay procedures, for example horseradish peroxidase. The kit may also contain standards, for examplespredetermined amounts of FF G-CSF (e.g. protein or RNA) may be labeledwith a detectable label. The kit may also contain disposable aspiratortips for use in extracting the oocytes and follicular fluid.

The kit may be used for the measurement of FF G-CSF for use in a methodof diagnosis, prognosis, and/or assisted fertilization treatment of afemale subject. The invention further provides the use of a reagent forthe detection of FF G-CSF for the prognosis of the likelihood ofestablishing pregnancy by assisted fertilization in a female subject.

The abovementioned antibodies, fragments and variants thereof, and othersuitable ligand-binding reagents, which may optionally be labeled with adetectable label, may be used for the manufacture of a diagnostic kitfor use in the treatment or diagnosis of suitability for assistedfertilization.

Levels of FF G-CSF may also be assayed via analysis of the levels of FFG-CSF mRNA present in samples obtained. In order to achieve this, FFG-CSF or fragments thereof may be used as a probe to determine levels ofG-CSF in the follicular fluid. Alternatively, levels of FF G-CSF may beestimated by analyzing the levels of FF G-CSF mRNA expressed in thefollicular fluid or in the granulosa cells. Granulosa cells around thecorona radiate may be stored at the stage of the decoronisation of eachoocytes and stored in RNA stabiliser (e.g. at 80° C.) until assay. Suchprobes may also be formulated into kits in a manner analogous to thosedescribed for antibodies, and may contain control nucleic acids. Probesfor the FF G-CSF gene may be designed for use as probes, for example foruse in a nucleic acid amplification assay.

EXAMPLES

The following non-limiting examples illustrate certain embodiments ofthe invention.

Example 1 Experimental Design

Patients

280 female patients presenting with infertility and included in an ICSIprogram were recruited between January 2005 to March 2007. The reasonfor inclusion in ICSI was predominantly male infertility but alsoprevious IVF failure or previous low fertilization rate in conventionalIVF. We proceeded to a randomization at the time of inclusion to notintroduce biases in the clinical patient selection. Each patient wasincluded one time within the study period. All patients were fullyinformed, and the Institutional Review Board approved this investigation(Comité Consultatif de Protection des personnes Poissy-St germain enLaye,).

Pre-Treatment

Patients underwent the classical procedure of ovarian hyperstimulation.We applied the protocol referred by their physician. Response tostimulation was controlled by serial blood tests and ultrasonicevaluation to control follicles and endometrial growth. Criteria fortriggering the ovulation was obtained when at least 5 follicles reached16 mm. The oocytes retrieval took place 35 to 36 hours after thetriggering of ovulation. The oocyte aspiration was performed undergeneral or local anesthesia with vaginal ultrasonography usingindividual 10 ml syringe for each follicle in the studied group. We thusadapted the classical method of oocyte aspiration in order toindividualize the follicular fluid of each oocyte collected.

Follicular Fluid Samples

The presence or absence of an oocyte in each follicle was immediatelyassessed and the follicles devoid of an oocyte were discarded. In thestudied group, individual follicular fluid sample, each corresponding toone mature oocyte was collected. The volume and the aspect (citrin,orange or hematic) of each follicular fluid sample were recorded.Individual follicular fluid samples were centrifuged and the supernatantaliquoted after proceeding to the anonymisation of each sample accordingto a database in order to blind subsequent analysis. Samples wereinitially stored at −20° C., then at −80° C. until assay. All theclinical and biological information were recorded in real-time on thedatabase (Medifirst).

Oocyte Fertilization and Embryo Culture Until Day 2

Oocytes were collected and cumuls and corona cells removed withhyluronidase 80 IU (Fertipro). Oocyte were injected in a 5 μl drop offlushing medium (JCD), with a sperm sample slowed by PVP medium(fertopto). Injected oocytes were cultured in singly 40 μl microdropletof ISM1 (Medicult, France) under oil at 37° C. Pronuclei number andaspect were assesses after 20 hours according to Gianaroli criteria. Onday 2, the number, fragmentation and regularity of each blastomere wererecorded. Embryo transfer was scheduled on day 2.

We divided the transferred embryos in two categories for analysis:

1. the best quality embryos defined by 4-5 cells on Day 2, 8-9 on Day 3,and less than 10% of fragmentation and regular cells (high qualityembryos), or

2. any other patterns (low quality embryos)

Only, the follicular fluids corresponding to the embryos transferredwere analysed using the Luminex method.

Evaluation of the Potential of Implantation

Each sample was related to a probability of implantation, described hereas the implantation rate. The clinical Implantation rate of an embryo isdefined for each tested sample as the number of yolk sac/number ofembryos transferred. Clinical implantation was defined at 8 weeks ofamenorrhea by the ultrasonic visualization of a yolk sac.

There is therefore three main categories in function of the outcome:

-   -   No implantation: Implantation Rate=0    -   Certain implantation: The number of embryos replaced was equal        to the number of yolk sac observed by ultrasound at 8 weeks of        amenorrhea (1 embryo replaced and single pregnancy, two embryos        replaced and twin pregnancy): Implantation Rate=1    -   Likely implantation which is a probability of implantation since        the number of yolk sac is lower than the number of embryos        replaced (e.g 1 out of 2, 1 out of 3, 2 out of 3, hence        Implantation Rate=0.5, 0.33, 0.66).

To construct the ROC curve (Receiver Operating Charateristics), we onlytook in account the two categories

-   -   No implantation,    -   Certain implantation: The number of embryos replaced was equal        to the number of yolk sac observed by ultrasound at 8 weeks of        amenorrhea (1 embryo replaced and single pregnancy, two embryos        replaced and twin pregnancy)

The discrimination attained between no implantation and certainimplantation in function of the concentration of G-CSF in each samplewas evaluated with ROC curve analysis (MedCalc Software, Mariakerke,Belgium). Sensitivity, specificity and the area under curve (AUC) ROCwere obtained for the three methods of detection. In a ROC curve thetrue positive rate (Sensitivity) is plotted in function of the falsepositive rate (100-Specificity) for different cut-off points. Each pointon the ROC plot represents a sensitivity/specificity pair correspondingto a particular decision threshold. A test with perfect discrimination(no overlap in the two distributions) has a ROC plot that passes throughthe upper left corner (100% sensitivity, 100% specificity). Therefore,the closer the ROC plot is to the upper left corner, the higher theoverall accuracy of the test (Zweig & Campbell, 1993). Calculation ofthe AUC-ROC provides the quantitative of accuracy, i.e. the ability ofG-CSF to discriminate between implantation and no implantation.

However, most of the embryos that implant were defined by a probabilityof implantation.

For example, if two embryos were transferred and only one implanted,each sample is characterized by a probability of 50% of implantation.

We thus defined for each method

-   -   Lower threshold of implantation defined by a negative predictive        value of 100% from the AUC-ROC curve.    -   Higher threshold of implantation defined by the highest positive        predictive value for implantation.        G-CSF Assays Performed on the FF Samples

Two Luminex methods of detection were successively applied forindividual follicular fluid samples from which transferred embryos wereissue.

From January, 2005 to June 2005

-   -   The Luminex technology was applied with the kit Biorad        (Hercules, Calif., USA, 17A11127, human cytokines, 27-plex kit).        From September 2005 to March 2007        80 of the samples were common to the previous Luminex biorad,        and 120 were new samples collected    -   The Luminex technology was applied with the R and D kit        (Minneapolis, Minn., USA, LUH000, LUH279, LUH270, LUH271,        LUH278, LUH208, LUH214, LUH215B, LUH285, LUH200, LUH280, LUH201,        LUH202, LUH204, LUH205, LUH206, LUH217, LUH317, LUH210, LUH293,        LUB000, LUB320, LUB294, LUB219, LUB213)

Multivariate and univariate analysis were performed. A p value below0.05 was considered as significant. Table 1 summarizes the populationand the number of samples analyzed successively using the two of themethods of investigation applied.

TABLE 1 Population and the number of samples analyzed successively usingtwo of the methods of investigation applied. Luminex Luminex Parametermeasured BIORAD R&D Number of patients included  71 121 Number ofindividual follicular fluids analysed 132 200 corresponding to antransferred embryo Mean Clinical Pregnancy Rate 31.5% 27.3% Meanimplantation rate   20%   18%

Evaluations using the Biorad® and R and D® Luminex kits are elaboratedin Examples 2 and 3.

Example 2 Evaluation Using the Luminex Kit Manufactured by Biorad®

132 follicular fluid samples corresponding to the subsequent 132transferred embryos were analyzed for levels of certain cytokines andchemokines. In particular, concentrations of IL-1beta, IL-1Ra, IL-2,IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IFNalpha, TNF-alpha, G-CSF, GM-CSF, VEGF, PDGF, FGF, IP-10, MCP-1, RANTES,EOTAXIN, MIP-1 alpha, MIP-1 beta were evaluated using Luminextechnology, utilizing a a Biorad® Luminex Kit.

The following results were obtained:

1) LIF, IL-1ra, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, G-CSF, VEGF,IP-10, MCP-1, Eotaxin and MIP-beta were detected in all the follicularfluid samples,

2) IL-1 beta, IL-5, IL-7, IL-17, TNF alpha, MIP-alpha were detected notdetected in any follicular fluid sample,

3) IL-15, GM-CSF, Rantes, PDGF, IFN-gamma, IL-9, IL-2, IL-15, FGF weredetected respectively in 95%, 94%, 88%, 81%, 76%, 65%, 60%, 48% and 22%of the follicular fluid samples.

G-CSF was not related to the embryo morphology if compared in regard thetwo categories best embryo quality versus others. Therefore, there wasno correlation between G-CSF and embryo morphological quality (Highquality versus other quality transferred embryos)

Cytokines, Growth Factors and Implantation Rates

Only one cytokine was associated in either univariate and multivariateanalysis with the potential of the corresponding embryo to implant,which was the Granulocyte-Colony Stimulating factor (G-CSF)

Embryos were Classified According to their Implantation Rates.

To construct the AUC-ROC for G-CSF, we took into account embryos thatdid not implant (n=89) and the ones that exhibited implantation (n=13).Certain implantation is defined when all the embryos replaced leads to ayolk sac.

The area under the ROC curve was 0.82 [0.73-0.89] and highly significant(p=0.0001) (FIG. 1). Thus, G-CSF is correlated with the implantationrate (r=0.40 p<0.0001).

We also found a significant difference between embryos with certainimplantation and no implantation (p=0.0002) and between embryos withcertain implantation and likely implantation (p=0.001) (Table 2)

TABLE 2 Correlation between implantation success and levels of FF G-CSFand FF IL-1ra measured using a Luminex kit manufactured by Biorad Numberof FF G-CSF +/− FF IL-1ra +/− embryos standard standard Implantationconcerned error (pg/ml) error (pg/ml) Certain implantation 13 25.3 +/− 1764 +/− 373 Likely implantation 30 21.6 +/− 1 225 +/− 106 Noimplantation 89   20.2 +/− 0.4 148 +/− 17 

According to the AUC-ROC curve, we defined a lower threshold and upperthreshold for G-CSF to evaluate if G-CSF concentration may be used toevaluate for each embryo a “potential of implantation” in order todecide the number of embryos we should replace.

The lower threshold was defined by the stronger negative predictivevalue of implantation. If G-CSF is lower than 20 pg/ml, negativepredictive value is at 100% from AUC-ROC. If G-CSF is over 24, positivepredictive value reaches its maximum: 40%

If all the embryos replaced are evaluated according to the level ofG-CSF, we can observe the subsequent differences of the implantationrate (Table 3).

TABLE 3 Correlation between implantation success and levels of FF G-CSFmeasured using a Luminex kit manufactured by Biorad; *p = 0.003 betweenmedium and low G-CSF; **p < 0.001 between high and low G-CSF Number ofembryos Mean implantation G-CSF (Luminex biorad) concerned rate LowG-CSF 45 9% (Below 20 pg/ml) Medium G-CSF 62 18%* (Between 20 to 24pg/ml) High G-CSF 25  44%** (Over 24 pg/ml)

Example 3 Evaluation of Follicular Fluids Using the Luminex KitManufactured by R and D

200 follicular fluid samples corresponding to the subsequent 200transferred embryos were analyzed. The concentrations of the followingcytokines and chemokines IL-1 alpha, IL-1 beta, IL-1Ra, IL-2, IL-4,IL-5, IL-6, IL-8, IL-10, IL-17, IFN alpha, TNF-alpha, G-CSF, GM-CSF,MIP-1 alpha, MIP-1 beta, RANTES, MCP-1, VEGF, were evaluated usingLuminex technology utilizing the kit made by R&D.

Detection of Cytokines and Chemokines

G-CSF, IL-1 Ra; IL-6, IL-8, MIP-beta, RANTES, MCP-1, VEGF were detectedin 95 to 100% of the follicular fluid samples tested:

-   -   IL-4, TNF-alpha, GM-CSF, IL-5 were detected in 75% to 94% of the        follicular fluid samples tested,    -   IL-1 alpha, IL-1 beta, IL-10, MIP-alpha were detected in 50 to        74% of the follicular fluid samples tested,    -   IL-2, IFN-gamma and IL-17 were detected in less than 50% of the        follicular fluid samples tested.        Cytokines, Chemokines and Implantation Rates

To construct the AUC-ROC for G-CSF, we took in account embryos that didnot implant (n=146) and the ones that certainly implanted (n=16) Thearea under the ROC curve was at 0.72 [0.65-0.79] and highly significant(p=0.0025) (FIG. 2)

A significant difference was observed for G-CSF between embryos withcertain implantation and no implantation (p=0.01) success and betweenembryos with likely implantation success and certain implantationsuccess (p=0.03) (Table 4)

TABLE 4 Correlation between implantation success and levels of FF G-CSFmeasured using a Luminex kit manufactured by R and D. Number of embryosG-CSF(pg/ml) +/− Implantation concerned standard error Certainimplantation 16   28 +/− 2.3 Likely implantation 37 20.5 +/− 1.7 Noimplantation 146 20.7 +/− 0.9

G-CSF was detected in all fluids and with low standard variations from asample to another which is a strong requirement to identify a biomarker.According to the AUC-ROC curve, we defined a lower threshold and upperthreshold for G-CSF to evaluate if G-CSF concentration may be used topredict for each embryo a “potential of implantation” that would help inthe decision of the number of embryos we should replace. The lowerthreshold was defined by the stronger negative predictive value ofimplantation. If G-CSF is lower than 15 pg/ml, negative predictive valuewas 100% from AUC-ROC. If G-CSF was over 34, positive predictive valuereaches it maximum of 27.8%.

If all the embryos replaced are evaluated according to level categorydefined by the AUC-ROC of G-CSF, we can observe the subsequentdifferences of the implantation potentiality (TABLE 5).

TABLE 5 Correlation between implantation success and levels of FF G-CSFmeasured using a Luminex kit manufactured by R and D; **p < 0.001between low, median, and high G-CSF Number of embryos Mean implantationG-CSF concerned rate Low G-CSF 61  9%** (Below 15 pg/ml) Medium G-CSF117 22%** (Between 15 to 34 pg/ml) High G-CSF 22 43.5%**   (Over 35pg/m)l

Example 4 The Mean of G-CSF in Pooled Follicular Fluid do not Reflectthe Variations Observed in Individual Follicular Fluids

Among 15 patients, all the follicular fluids that lead to an embryoindependently of its outcome were evaluated in the same cohort. 76samples were evaluated using the Luminex kit manufactured by Biorad.

For each sample, we evaluated the following ratio. Mean G-CSF in pooledFollicular fluid Less G-CSF concentration in individual FF (n=76).

FIG. 3 is graph showing the variation in concentration of individualfollicular fluid of a same cohort of embryos obtained from 10 multiplesubjects. Each box shows the variation of individual follicular fluidsfrom the mean in a same cohort of embryos generated. These observationsuggests that all the embryos generated are not equal in regard ofFF-GCSF and hence in their potential of implantation.

What is claimed is:
 1. A kit for determining for a female subject theimplantation potential of embryos obtained or to be obtained by assistedfertilization comprising: system for measuring for a plurality ofoocytes collected from said subject the level of follicular fluidgranulocyte-colony stimulating factor (G-CSF) present in the follicularfluid (FF) of a follicle of each collected oocyte, which systemcomprises at least one reagent suitable for detection of levels of FFG-CSF or FF G-CSF mRNA, and a plurality of disposable aspirator tips forremoving an oocyte and follicular fluid from a subject.
 2. Kit accordingclaim 1, further comprising a set of concentration standards of G-CSF.3. Kit according claim 2, wherein the concentration standards arecommercial G-CSF standards.
 4. Kit according to claim 1, wherein the atleast one reagent comprises an anti-G-CSF antibody for detection oflevels of FF G-CSF.
 5. Kit according to claim 4, wherein the anti-G-CSFantibody is labeled.
 6. Kit according to claim 1, wherein the reagent isa nucleic acid probe for detection of levels of G-CSF mRNA.
 7. Kitaccording to claim 6, wherein the nucleic acid probe is labeled.